INTRODUCTION: The periodontal ligament (PDL) is a structure between the alveolar bone and cementum, essential for tooth stability and composed of diverse cell types. Mohawk homeobox (Mkx) is a master transcription factor that regulates tendon and ligament homeostasis. However, the specific cell populations expressing Mkx and its role in mechanotransduction during orthodontic tooth movement (OTM) remain unclear. MATERIALS AND METHODS: We conducted single-cell RNA sequencing on wild-type rat PDL at 0 day, 1 week, and 2 weeks of post-OTM using coil springs to elucidate Mkx's function and the changes in cell populations under continuous mechanical stimulation. In addition, RT-qPCR was performed to assess the relationship between tenogenic gene expression and Mkx expression in human PDL cells. RESULTS: The rat PDL was identified to consist of 14 clusters, with Mkx and Scleraxis (Scx) expressed in distinct cell populations. Collagen and ECM production increased throughout the OTM period, while the sterile inflammatory response was initially heightened and later diminished, indicating that bone remodeling occurs later in the inflammatory response. Overexpression of MKX in human PDL cells enhanced COL1A1 and DECORIN expression. CONCLUSION: Mechanical stimulation of the PDL appears to trigger an aseptic inflammatory response that disrupts PDL homeostasis and promotes bone remodeling. Mkx may exert a protective effect on the PDL during mechanical stimulation.